This laboratory is identifying extracellular factors that determine functional receptor expression in tissues that are capable of diverse pharmacological responses. Smooth muscle cells, for example, have the capacity to express a wide variety of neurotransmitter/hormone receptor types. This proposal directly addresses the molecular nature of extracellular signals that regulate the pharmacological responsiveness of smooth muscle cells and the mechanisms by which this regulation is mediated. This laboratory has previously established that the sensitivity of cultured smooth muscle cells to the neuropeptide, substance P, is completely and specifically suppressed by factors associated with the extracellular matrix (ECM). Furthermore, it is now established that this suppression of substance P responsiveness is mediated by an integrin receptor, and is mimicked uniquely by an ECM and smooth muscle-associated molecule, thrombospondin. The main thrust of this proposal centers on rigorously establishing whether thrombospondin is the molecule responsible for the ECM effect, and identifying the specific integrin receptors involved. These goals will be achieved primarily by utilizing recombinant thrombospondin and specific antibodies against thrombospondin, as well as, antibodies to the various integrin receptors. In all of these studies, the dramatic induction of sub-stance P responsiveness in cultured smooth muscle cells will be used as the assay for activity. In addition, the mechanism by which the cells become sensitive to substance P will be addressed by receptor binding studies using radiolabeled substance P. If increased receptors are evident in these cultured cells, then cDNA probes for the mRNA of the substance P receptor will be utilized to determine whether transcriptional regulation of receptor expression can account for the increased substance P responsiveness. An understanding of the molecules that regulate cellular responsiveness to different hormones and neurotransmitters may allow rational interventions in a clinical setting. One of the long range goals on a clinical level is to devise methods to specifically instruct tissues to alter the expression of the receptors that are involved in a disease state. Regardless of whether the receptors were actively involved in the etiology of the disease, such alterations in receptor expression could effectively ameliorate painful or dangerous symptoms. For example, several lines of evidence indicate that the substance P receptor is involved in certain aspects of pain perception. Control of receptor expression is related, of course, to the basic strategy of treating disease and disease symptoms with drugs. However, the control of functional receptor expression offers an alternative to classical pharmacology and may prove to be more tissue specific and/or more chronically effective than receptor blockade achieved pharmacologically. For example, antagonist blockade of receptors often results in up-regulation of that receptor type, producing tolerance to the antagonist and the necessity of increased drug dosages and, often, concomitant toxicity. By affecting the very mechanisms by which cells determine their receptor expression, the homeostatic responses of cells may be circumvented.